The Polymerase Chain Reaction (PCR) (Mullis and Faloona 1987; Saiki, Gelfand et al. 1988) is an integral technique in scientific research. Cloning of PCR products is often an obligate step towards reaching a research objective. PCR-cloning presents numerous challenges and various techniques have been devised over the years to minimize its limitations. Cloning of PCR products generally fall into one of the following methodologies; i) traditional PCR-cloning using restriction enzymes and ligase, ii) T-vector or TA-cloning, iii) uracil DNA glycosylase (UDG)-based cloning iv) PCR-based techniques, v) in vivo recombinase methods, and vi) exonuclease-mediated cloning. Each of these methodologies is described below.
i) Traditional PCR-Cloning
Cloning of PCR-amplified DNA was traditionally facilitated by incorporating restriction endonuclease (RE) sites into the PCR primers, allowing for subsequent digestion of the PCR product with the appropriate enzyme followed by insertion into a compatible vector (Sharf et al. 1990). One problem often encountered with this method is that REs are notoriously poor cutters when their recognition sequences are close to the ends of the DNA substrate (Kaufman and Evans 1990). A second complication is that sequences within the PCR-amplified region can be lost if a second restriction site for the same RE is unknowingly present. This necessitates that the complete sequence of the PCR product be known prior to choosing which RE to use. Once the vector and DNA insert have been digested with the same RE, the two DNA molecules can be covalently joined by DNA ligase in a reaction typically taking 4-12 hr. It should be noted that during any ligation reaction, it is critical that the vector and insert are present in appropriate ratios, which is often difficult to determine. The reaction products are then used to transform competent E. Coli cells. A subtle variation on this theme called “ligation by overlap extension” has been devised which does not require any subsequent ligation reaction, but does require two additional primers, and the entire vector sequence itself must be amplified (Shuldiner, Tanner et al. 1991).
Blunt-end cloning of PCR fragments has also been used extensively, although this technique is relatively inefficient because of the problems encountered by DNA ligase when joining together two blunt-ended DNA molecules. This technique is also complicated by the fact that Taq polymerase (the prototypical PCR amplifying enzyme) has a propensity for adding 3′ terminal adenine residues through its terminal transferase activity (Clark 1988; Mole, Iggo et al. 1989). Approximately 50% of all PCR products generated using Taq polymerase contain these 3′extensions (Clark, 1988). One way around this problem is to “polish” or remove these added adenine residues with Klenow (Hemsley, Arnheim et al. 1989) or T4 DNA polymerase (Costa and Weiner 1994), adding an additional step to the protocol.
Recent discoveries of different thermostable polymerases including Pfu polymerase from Pyrococcus furiosis and VentR polymerase from the archaebacteria Thermococcus litoralis do not produce 3′ adenine residue extensions due to inherent 3′-5′ exonuclease activities. In addition to the problems produced by the amplifying polymerase, this technique does not allow for directional cloning, meaning that the orientation of insert in the recombinant DNA cannot be predetermined.
ii) T-Vector and TA-cloning
The terminal transferase activity of Taq polymerase has been exploited by many researchers in a technique now commonly known as TA-cloning. The chosen vector is digested with the appropriate RE so as to yield ends with protruding thymidine residues, the natural complement to the 3′-overhanging adenosine residues found on the PCR-amplified DNA. The most significant drawback of this technique is that vectors must be specifically engineered to produce compatible ends. The only simple way of accomplishing this goal is to restrict the vector to produce blunt ends, and then treat it with Taq polymerase in the presence of only dTTP's. Numerous companies have developed kits based on this technique, including pCR-Script™ SK(S) kit from Stratagene, pGEM®-T from Promega, the SureClone™ ligation kit from Pharmacia and the pT7Blue T-vector kit from Novagen. The main limitation to these methods is that a vector supplied by the manufacturer must be used and a second subcloning step is often necessary to move the cloned DNA fragment into a vector of choice. An inexpensive alternative to buying these kits is to use a T-vector pGEM®-5fZ(+) which is available for little or no cost from the American Type Culture Collection (ATCC). This vector when digested with XcmI provides the T-overhangs used for TA-cloning (Kovalic, Kwak et al. 1991; Mead, Pey et al. 1991). Numerous other T-vectors have been developed independently (Cha, Bishai et al. 1993; Ichihara and Kurosawa 1993) which, after appropriate RE digestion, yield appropriate ends. Such vectors, however, required extensive manipulations to create. Some other potential problems with these kits have been reported recently (Hengen 1995). High backgrounds were observed for the pCR-Script vector when tested alone, PCRII contains a pBR322 origin of replication and thus replicate to low copy numbers, and repeated freeze-thaw cycles at −20° C. can lead to instability and loss of the T-tails. All the T-vector techniques suffer from the drawback that they are non-directional and require a ligation step.
Invitrogen have improved upon traditional TA-cloning by bypassing the need for a ligation step. This method, called TOPO TA-cloning, takes advantage of a reaction catalyzed by a vaccinia virus enzyme called topoisomerase I (Shuman, 1994 and U.S. Pat. No. 5,766,891). Topoisomerase can bind to double-stranded DNA and cleave the phosphodiester backbone in one of the duplex strands. The enzyme is sequence specific, cleaving primarily at the recognition sequence 5′-(C/T)CCTT↓-3′ (Shuman and Prescott 1990; Shuman 1991). The enzyme is capable of re-ligating the original strand back together, or ligating two heterologous DNAs in the formation of a recombinant species (Shuman 1992; Shuman 1992). The reaction is very efficient requiring only a 5 minute benchtop incubation. The methodology also has advantages which obviates the need for ligase, does not require knowledge of the entire insert sequence and no additional nucleotides need be added to PCR primers. However, only specific plasmids engineered to contain the TOPO recognition sequence can be used. These vectors are produced by restricting the vector followed by adding specific linkers or adaptors, which is not a trivial task. Another limitation of this technique, is that the TOPO recognition sequence must be located within 10 bp from the 3′-ends of the vector, and furthermore, the insert must have a 5-OH group. The issue has been raised that internal recognition sequences within the amplified DNA may result in complications, however these sites are simply religated and do not impose any restrictions on this technique (Shuman 1994; Stivers, Shuman et al. 1994). Under general use, the Invitrogen kit provided another potential problem (unpublished results). The traditional method for screening clones, called blue-white selection, does not produce definitive results with the Invitrogen kit. Therefore, it is necessary to assay both white and light-blue colonies to ensure the correct construct is obtained.
iii) Uracil DNA-Glycosylase (UDG)-Based Cloning
Rashtchian et al. (1992) developed a ligase independent PCR cloning method using uracil DNA glycosylase (UDG), an enzyme whose normal cellular role is a DNA repair enzyme. The technique requires a 12-bp addition (CUACUACUACUA) to the 5′end of the PCR primers. The glycosylase selectively removes dUMP residues at the ends of the PCR products which disrupts proper base-pairing leading to single-strand 3′-overhangs (Duncan and Chambers 1984; Longo, Berninger et al. 1990). These 3′-overhangs can anneal to appropriately prepared single-strand ends of a vector. Uracil glycosylase is not active with thymine residues, the DNA counterpart of uracil residues (Duncan and Chambers, 1984), and is capable of removing dUMP residues even near the extreme ends (Varshney and van de Sande 1991). This methodology requires that the vector contain the appropriate complementary sequences, and is not amenable to use with proofreading polymerases such as Pfu or VentR polymerases (Sakaguchi, Sedlak et al. 1996). The researchers must therefore use Taq polymerase which has a significantly increased error frequency. UDG-based cloning has been commercialized by Life Technologies with their Clone AmpR pUC system.
A variation on UDG-cloning takes advantage of the abasic sites (AP) produced by UDG-cleavage at dUMP residues. These AP sites are substrates for AP endonucleases such as T4 endonuclease V or human AP endonuclease I. Treatment with either of these repair enzymes yields a 5-P which is suitable for subsequent ligation into the appropriate vector. One drawback of this method is the requirement for a modified base (deoxyuridine) in the primer, and success relies on two enzymes in addition to ligase treatment. A second more obscure variation of UDG-cloning involves the use of a non-base residue called 1,3-propanediol in a predetermined position within the PCR primer, which can yield compatible 5′-ends for cloning, however, this method is much less efficient than other ligase-independent cloning methods (Kaluz and Flint 1994).
iv) PCR-Directed Cloning
PCR-specific cloning methods are often one-step procedures in which the recombinant DNA is produced during the amplification procedure itself. There are many variations on this theme, in which some are ligase-dependent and others are not. These methods are primarily used to produce site-specific mutations in cloned genes. A brief description of the current techniques follows.
Ligase-Dependent Methods
a) Stratagene have commercialized a technique for the cloning of blunt-end PCR fragments (Weiner 1993), originally described by Liu and Schwartz (1992). Their methodology requires phosphorylating the 5′ end of the PCR primers. The recipient vector is linearized and treated with calf intestinal alkaline phosphatase (CIAP) and then digested with a second restriction enzyme to yield compatible ends. This is a rather convoluted technique but the resultant vector is mono-phosphorylated and allows for directional cloning. They reported a 95% success rate for directional cloning, however their technique requires an ethanol precipitation and still relies on the actions of ligase.
b) “Hetero-stagger cloning” is another ligase-dependent method which requires a total of four PCR primers (Liu 1996). One set of primers is the traditional PCR primer pair and the second set is equivalent to the first, but includes three additional 5′-GGG residues. The DNA is amplified under normal PCR conditions, the products are denatured by heat and then allowed to reanneal slowly by cooling. Reannealing results in the formation of four distinct species. Only 50% of the products are theoretically cloneable, and only 25% of the products would successfully result in directional clones. The only claimed advantage to this technique is that it allows for modern proofreading polymerases to be used during amplification.
c) A variation of the staggered re-annealing technique has also been used which requires only one primer pair (Ailenberg and Silverman 1996).
d) More recently, Gal et al. (1999) have devised a technique called “autosticky PCR” (AS-PCR) (patent application HU9801320). This technique takes advantage of the observation that abasic sites present in DNA can stall DNA polymerases. In this method, PCR primers are designed to contain abasic sites, which stall the amplifying polymerase, resulting in 5′-overhangs thus enabling ligation into a suitably digested vector. The abasic site is produced by the incorporation of tetrahydrofuran, a stable structural analogue of 2-deoxyribose, at the desired position. This method does provide for directional-cloning, but requires non-traditional reagents and an overnight ligation is recommended.
Ligase-Independent Cloning Methods (LIC)
a) The original ligase PCR-cloning method was described by Shuldiner et al. (1991). Since then, numerous adaptations of this technique have been developed. The technique described here (Temesgen and Eschrich 1996) requires three PCR primers, in which one of the primers contains an additional 24 nucleotides. This process involves two distinct PCR amplifications, thus increasing the probability of introducing PCR errors into the products. However, the linear products can be directly transformed into E. coli obviating the need for ligase. Competent E. coli strain TG2 cells are required, and it is unclear if classical strains such as JM105 or DH5α are able to be substituted. This technique does provide for directional cloning, although the success is related to the PCR parameters in the second PCR step. Any vector can be used in the technique and no restriction enzymes are needed.
b) Garces and Laborda (1995) reported a similar technique only requiring two PCR primers, one of which has a 20 bp 5′-extension. The reaction occurs within a single-tube reaction, and can be adapted for use with any vector, but the efficiency is greatly affected by the PCR parameters.
v) In Vivo Recombination-Based Cloning
PCR-cloning is traditionally completed within the test-tube environment of the laboratory, however, there are at least two reports of cloning using in vivo systems. The following technique was based on the observation that when yeast were co-transfected with a linear template and a gapped plasmid, homologous recombination was able to “patch” the two species together (Guthrie and Fink, 1991). PCR products have since been cloned in yeast using this method (Scharer and Iggo 1992). A similar phenomenon has been reported in E. coli (Oliner, Kinzler et al. 1993). This technique presumably takes advantage of endogenous exonuclease or polymerase activities encoded by the host, but there is no speculation as to what is exactly occurring. The PCR primers are designed to contain 5′-sequences which are identical to sequences adjacent to a chosen RE site. The linearized vector and the PCR products are co-transfected into E. coli strain JC8679. This technique may not be suitable for use with traditional E. coli strains because independent reports indicate that DH5α cells cannot catalyze intramolecular gap repair, and thus might not be expected to catalyze inter-molecular recombination (Hanahan, 1985). A similar methodology described by Bubeck et al. (1993) reported successful recombination in DH5α cells but only if they were transformed by CaCl2 methods. Two more commonly used techniques for bacterial transformation known as heat shock and electroporation were unsuccessfully used in the above experiment.
vi) Exonuclease-Based PCR Cloning
A completely different approach to the cloning of PCR fragments involves the generation of single-strand overhangs through the action of various exonucleases. All of the exo-based methods are ligase-independent and are based on the technique originally reported by Aslanidis and deJong, (1990). Numerous modifications to this technique have allowed for improvements in the method (Haun, Serventi et al. 1992; Kuijper, Wiren et al. 1992), both of which use the 3′-5′ exonuclease activity of T4 DNA polymerase. PCR primers are designed to contain a 5′-extension complementary to sequences adjacent to a chosen RE site within the vector. Single-strand overhangs are generated through the exonucleolytic digestion by T4 pol and annealing of single-strand regions between the vector and insert is sufficiently stable to allow for direct bacterial transformation. These techniques require delicate control of incubation periods, as these enzymes are extremely efficient, and if one is not careful, excess DNA can be digested. Kuijper et al. (1992) also reported that there is great variation between enzyme preparations, therefore, requiring fine-tuning of temporal conditions with each new batch of enzyme. A second drawback to these specific methods is the requirement for the addition of dTTPs or dATPs in the exo reaction to stop the enzyme at the appropriate positions.
A similar method to that reported above uses a different enzyme called exonuclease III (Hsiao 1993), which was originally used for cloning in 1992 (Kaluz, Kolble et al. 1992). Its limitation is that only blunt-ended or 5′-overhanging substrates can be efficiently cloned. Substrates with 3′-overhangs cannot be cloned by this method.
More recently, phage T7 Gene6 exonuclease has been used for PCR-cloning (Zhou and Hatahet 1995). In this technique PCR primers are designed to include internal phophorothioate bonds positioned towards the center of the primers. The 3′end of the primers are standard PCR primers, whereas the 5′ ends are designed to be complementary to sequences adjacent to a certain RE site. This method produces directional clones and is ligase-independent but requires the use of non-standard PCR primers. U.S. Pat. No. 5,580,759 (Yang, et al. also discloses a method of construction of recombinant DNA by exonuclease recession.
In summary, a wide variety of methods exist for the cloning of PCR products, and each has its advantages and disadvantages. There remains a need for an optimal cloning method having the following characteristics:                compatible with the use of any vector and any restriction enzyme;        requires only two PCR primers comprised solely of natural bases;        ligase independent;        time efficient;        provides almost exclusively directional cloning;        only the terminal sequences of the amplified region need to be known;        no possibility of internal digestion of the PCR product;        any type of amplifying polymerase can be used;        compatible with various readily available E. coli strains;        transformation of bacteria can be accomplished through a variety of techniques;        unambiguous selection; and        adaptable to other techniques such as combinatorial cloning.        